Attempts at Cloning pBR322Δrop

In order to study the effect that the Rop protein may play in the observed exclusion of pBR322 by pUC19, attempts to cut out the rop gene from pBR322 were made. Several different pairs of restriction enzymes were tried for the digestion, and the innate ligation ability of E. coli cells was used to recircularize the cut plasmids. Transformed cells were screened using both ampicillin and tetracycline plates, and attempts to isolate pBR322 ∆ rop through mini-prep or slot-lysis were performed on promising colonies. In the end, all attempts to clone pBR322 ∆ rop failed. This paper describes the methods used as well as putative reasons for why the techniques failed. The main problems were non-optimal digest conditions and screening of inadequate numbers of colonies.